Extended Data Fig. 6: Chemogenetic stimulation of SDH astrocytes and Hes5-negative astrocytes in deeper laminae of the SDH.
a, Schematic illustration of the experimental protocol. AAV-hM3DqFLEX was microinjected unilaterally into the SDH of Gfap-Cre mice. b, Immunofluorescence of hM3Dq (HA-tag) in the SDH of Gfap-Cre and wild-type mice injected with AAV-hM3DqFLEX. Note that hM3Dq expression was induced only in Gfap-Cre;AAV-hM3DqFLEX mice and that its localization was observed in the SDH, including superficial laminae. c, Immunohistochemical identification of hM3Dq (HA-tag)-expressing cells using cell-type markers (SOX9, GFAP, NeuN, IBA1 and APC) in the SDH of Gfap-Cre;AAV-hM3DqFLEX mice. Arrows indicate hM3Dq-positive cells. hM3Dq was selectively expressed in astrocytes. The data in b and c were representative of three independent experiments. d, hM3Dq-induced mechanical hypersensitivity in Gfap-Cre;AAV-hM3DqFLEX mice (saline, n = 4 mice; CNO, n = 5 mice); repeated measures two-way ANOVA with Bonferroni’s multiple comparison test and Kruskal-Wallis test with Dunn’s multiple comparisons test. The letter indicates the following P value versus the ipsilateral side of the saline group (a, P < 0.0001). e, Schematic illustration of the experimental protocol and timeline in Hes5-CreERT2 mice using the Cre-Off system. AAV-gfaABC1D-hM3DqFLEX was microinjected unilaterally into the SDH of Hes5-CreERT2 mice, in which the hM3Dq sequence can be inverted by tamoxifen to stop hM3Dq expression in Cre-positive (Hes5+) cells. By contrast, Cre-negative (Hes5-negative) astrocytes (located in the deeper laminae) express hM3Dq. f, c-Fos+ neurons in laminae I–IIi and III–V of vehicle or CNO-treated Gfap-Cre;AAV-hM3DqFLEX mice with or without Aβ fiber stimulation (vehicle without stimuli, n = 6 mice; CNO without stimuli, n = 5 mice; vehicle with stimuli, n = 6 mice; CNO with stimuli, n = 6 mice; one-way ANOVA with Tukey’s multiple comparisons test). Insets: c-Fos immunofluorescence in laminae I–IIi. Scale bars, 50 μm (c), 100 μm (f insets) and 200 μm (b, f). Dashed lines (b, f) indicate the boundary between the gray and white matters. Data show the mean ± s.e.m. See source data for statistical parameters.
